Abstract
Although LINE-1 (L1) sequences constitute the most important family of retrotransposons in the human genome, their transcriptional regulation is poorly understood. Specifically, their unusual internal promoter is incompletely characterized. Current promoter prediction programs fail to identify the promoter in the 5′UTR of the active LINE-1 element L1.2B. Experimental investigation of this promoter using reporter gene assays in various human and murine cell types confirmed that the promoter consists of two segments, and demonstrated that the distal portion is essential for cell-type-independent activity. No differences in promoter activity were found between normal and transformed cells. The complete promoter was shown to possess ≈20% of the activity of the strong early promoter of cytomegalovirus, and to be capable of directing the expression of levels of p53 sufficient to kill normal and transformed human cells. Thus, active LINE-1 elements contain highly active promoters capable of driving cell-type-independent expression, which are of potential use in mammalian expression constructs. In vitro methylation of the promoter at Hpa II sites decreased its activity independently of cell type, but this repression was alleviated in MBD2−/− cells. Surprisingly, mutation of specific Hpa II sites was also found to reduce promoter activity. Thus, efficient repression of the L1.2B promoter by DNA methylation may involve MBD2 binding, but at least one Hpa II site also appears to be involved specifically in transcriptional activation. Since neither promoter activity nor the efficiency of repression by methylation differed between normal and tumor cells, the re-activation of LINE-1 sequences observed in tumor cells is probably caused by hypomethylation of the promoter.
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Acknowledgements
We thank Andrea Prior for technical assistance and Dr. Gaby Reichmann for help with the FACS analysis. We are grateful to Prof. Brian Hendrich (Edinburgh University) for providing MBD2 knock out cells and the control cell line MEF, and for helpful discussions. This work was supported through funding by the Forschungskommission der Medizinischen Fakultät der HHU and a fellowship awarded by the German National Scholarship Foundation to CS.
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Communicated by C. P. Hollenberg
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Steinhoff, C., Schulz, W.A. Transcriptional regulation of the human LINE-1 retrotransposon L1.2B. Mol Genet Genomics 270, 394–402 (2004). https://doi.org/10.1007/s00438-003-0931-2
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DOI: https://doi.org/10.1007/s00438-003-0931-2